Structural basis for Sarbecovirus ORF6 mediated blockage of nucleocytoplasmic transport

The emergence of heavily mutated SARS-CoV-2 variants of concern (VOCs) place the international community on high alert. In addition to numerous mutations that map in the spike protein of VOCs, expression of the viral accessory proteins ORF6 and ORF9b also elevate; both are potent interferon antagonists. Here, we present the crystal structures of Rae1-Nup98 in complex with the C-terminal tails (CTT) of SARS-CoV-2 and SARS-CoV ORF6 to 2.85 Å and 2.39 Å resolution, respectively. An invariant methionine (M) 58 residue of ORF6 CTT extends its side chain into a hydrophobic cavity in the Rae1 mRNA binding groove, resembling a bolt-fitting-hole; acidic residues flanking M58 form salt-bridges with Rae1. Our mutagenesis studies identify key residues of ORF6 important for its interaction with Rae1-Nup98 in vitro and in cells, of which M58 is irreplaceable. Furthermore, we show that ORF6-mediated blockade of mRNA and STAT1 nucleocytoplasmic transport correlate with the binding affinity between ORF6 and Rae1-Nup98. Finally, binding of ORF6 to Rae1-Nup98 is linked to ORF6-induced interferon antagonism. Taken together, this study reveals the molecular basis for the antagonistic function of Sarbecovirus ORF6, and implies a strategy of using ORF6 CTT-derived peptides for immunosuppressive drug development.

argument about binding affinity influencing strength of repression. one possible route to explore this further would be to just express the CTT or NTE of MHV68/VSV M (or make a chimeric orf6 Nterminal/MHV68 CTT hybrid) and see if these proteins cause less of a reduction of GFP expression compared to orf6.
-would also like to see MHV68/VSV M on the blot in fig 4A. -would also like to see the authors add more interpretation of results in the discussion regarding the following..1) speak to how orf6 is capable of blocking stat1 nuclear import from their structure 2) make it more quantitative (e.g., confocal images) to be better able to see if binding affinity also correlates with strength of nuclear import blockade 3) discuss whether the orientation of the viral protein-RAE1 ( fig 2E) influences ability to block nuclear import. rather than fig 4B-D, a more direct measure of binding affinity vs RNA export would be more interesting. specifically, i'm thinking if they could look at degree of RNA dislocation from the rae1/nup98 complex across their different constructs (something like fig 3 in https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4078809/), that could better make their argument of binding affinities stronger and more directly connected to mechanism.

Point-to-point responses to reviewers' comments
Reviewer #1 (Remarks to the Author): The manuscript by Gao et al reports the structural basis for the recognition of Rae1-Nup98 by SARS-CoV and SARS-CoV-2 Orf6 protein. Orf6 is a key interferon antagonist, and it has been implicated in blocking nuclear import of STAT as well as nuclear export of mRNA. The crystal structures of Orf6-Rae1-Nup98 reported here reveal the molecular details for the interaction between Orf6 and Rae1-Nup98. The authors examined the contribution of individual Orf6 residues to Rae1-Nup98 binding affinity and the disruption of mRNA nuclear export as well as STAT1 nuclear import. The results showed that Met58 is critical for Orf6's inhibitory functions, which is confirmatory of published studies. Several Orf6 residues flanking Met58 also contribute to Rae1-Nup98 recognition. Overall, this work provides an important piece of information on how Orf6 targets Rae1-Nup98, but does not significantly extend the current working model of Orf6-mediated activities.

Response:
We thank this reviewer for the comments. In this study, we provide a structural basis for the hijacking of the cellular nucleocytoplasmic transport machinery by two important Sarbecovirus ORF6 proteins, and identify key residues of ORF6 CTT that determine binding affinity of ORF6 to Rae1-Nup98. Furthermore, we demonstrate that the binding affinity of ORF6 to Rae1-Nup98 is relevant to its role in nucleocytoplasmic trafficking blockade and IFNs suppression. Additionally, in the revised manuscript, we added new results showing a correlation of ORF6/Rae1-Nup98 binding affinity with RNA disassociation from the Rae1-Nup98 complex. By combining these findings, we propose a strategy for using ORF6 CTT peptide as a potential immunosuppression agent.
Here are some specific points. 1. The statement "P57 governs the orientation of binding" in the abstract (detailed description in page 6) is incorrect. The N' and C' of VSV M protein are incorrectly labeled in Fig 2E. In fact, Orf6, MHV-68 Orf10, and VSV M bind to Rae1 in the same N' to C' orientation.

Response:
We apologize for the incorrect description and the labelling in Fig 2E. We corrected these sentences in main text (page 6, line 88-91) and the labeling in the revised Fig 2E (see revised Fig 2e) Figure 3B to 3T, dissociation constants are all labeled as kD in the figure and the legend. Line 28: "the C-terminal (CTT) of SARS-CoV-2 ….". Orf6 is missing. Line 349: The complexes of protein:peptide were reconstituted by incubating protein and peptide were mixed at…" Line 557: "E-H. ITC titrations of between…."

Response:
Our revised manuscript has been proofread and corrected by a native English speaker. All  This manuscripts offers structures and binding affinities of the CTT of SARS-CoV and SARS-CoV-2 orf6 with the Rae1-Nup98 complex, an important target of this accessory gene in the sarbecoviruses. The authors recover additional orf6 mutations associated with increased and decreased nucleocytoplasmic trafficking. Many of the other assays are standard and confirmatory for prior work that has been done on the orf6-rae1-nup98 complex. The main addition is the structure which has only been imputed to date as well as the direct binding assays and estimations of Kd, which are both commendable and of significance to the field.

Response:
We thank this reviewer for summarizing the key contributions of our work and positive comments.
Minor comments.
-axis in Figure 3A should be labeled.

Response:
We added x axis label (folds) in the revised Fig. 3a, and provided details of calculating this value in the legends (page 37, line 2-3).
-the reduced affinity of MHV68 and VSV M compared to SARS/SARS2 takes away from the overall argument about binding affinity influencing strength of repression. one possible route to explore this further would be to just express the CTT or NTE of MHV68/VSV M (or make a chimeric orf6 N-terminal/MHV68 CTT hybrid) and see if these proteins cause less of a reduction of GFP expression compared to orf6.

Response:
We carried out new experiments and added more results to the revised manuscript. The full length MHV68 ORF10 and VSV M actually exhibited a stronger interaction with Rae1-Nup98 than that of full length Sarbecovirus ORF6 (revised Fig 4a) -would also like to see MHV68/VSV M on the blot in fig 4A.

Response:
We thank the reviewer for the suggestion. We included MHV-68 ORF10 and VSV M on the western blotting in revised Fig. 4a.
-would also like to see the authors add more interpretation of results in the discussion regarding the following..1) speak to how orf6 is capable of blocking stat1 nuclear import from their structure 2) make it more quantitative (e.g., confocal images) to be better able to see if binding affinity also correlates with strength of nuclear import blockade 3) discuss whether the orientation of the viral protein-RAE1 ( fig 2E) influences ability to block nuclear import.